Previous reports have indicated that the simian retrovirus (SRV) serogroup 3 genome contains a 219 bp sequence, extending from the 3' intergenic nontranslated region into the 3' LTR, that can serve as a cis-acting element to promote rev-independent human immunodeficiency virus (HIV) replication. This sequence, containing a constitutive transport element (CTE), enables the transport of unspliced HIV RNA from the nucleus to the cytoplasm, in the absence of rev and the rev-responsive element (RRE). To identify potential cellular protein factor(s) that bind the CTE region in the related D2/RHE/OR/V1 genome, we conducted RNA mobility shift assays using nuclear extracts prepared from uninfected and D2/RHE/OR/V1-infected Raji cells. Using polymerase chain reaction (PCR), we generated fragments representing three sub-regions of the CTE region from the D2/RHE/OR/V1 intergenic spacer, for individual subcloning of fragments into pGEM3Zf(+). Sense RNAs were synthesized by in vitro transcription. RNA mobility shift experiments, using both homologous and heterologous sub-region competitors, indicate that R1, R2, and R3 RNAs bind a common host-encoded protein, with R1 and R3 RNAs binding at non-overlapping sites on the same protein molecule. In addition, heterologous competition analyses also indicate the potential existence of a second protein bound to R2 RNA. Similar results were obtained using nuclear extracts from uninfected or D2/RHE/OR/V1-infected Raji cells. To determine the functional significance of this intergenic region to virus production, we deleted the intergenic spacer from an infectious molecular clone of D2/RHE/OR/V1. Transfectants containing the D2/RHE/OR/V1 molecular clone released virus into the culture medium within 4 - 5 days post-transfection; no reverse transcriptase activity was observed in culture medium obtained from transfectants containing the mutant recombinant, even at 5 days post-transfection. Thus, preliminary evidence indicates that the presence of the intergenic nontranslated region may play an important role in simian retrovirus particle formation and/or release.